CN114306276A - Preparation method of multifunctional intestinal tract diagnosis and treatment preparation and product thereof - Google Patents
Preparation method of multifunctional intestinal tract diagnosis and treatment preparation and product thereof Download PDFInfo
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Abstract
The invention provides a preparation method of a multifunctional intestinal tract diagnosis and treatment preparation and a product thereof, wherein the multifunctional intestinal tract diagnosis and treatment preparation product simultaneously has a multifunctional probe for PET imaging, ultrasonic imaging and photothermal therapy, can simultaneously meet the requirements of accurate tumor positioning and high flexibilitySensitive long-period imaging is carried out, and the targeting imaging efficiency of the probe to the tumor is improved. Macrophages are used as a drug carrier 'reservoir'. The enrichment of the carrier at the tumor part is enhanced through the transfer effect of macrophage, and the chitosan can be effectively adsorbed on the surface of intestinal mucosa.18Class F ribozymes act as imaging agents for PET. The tumor position and the metastasis are determined through PET imaging, and the accurate positioning of the whole tumor is realized. Fluorocarbon compounds are used as ultrasound contrast agents. The diagnostic capability of local tumors is improved through ultrasonic imaging. The gold nanorods have photothermal effect and have the effect of treating tumor parts.
Description
Technical Field
The invention relates to a preparation method of a multifunctional intestinal diagnosis and treatment preparation and a product thereof, in particular to a preparation containing18F-fluorocarbon, M2 type macrophage probe of gold nanorod and preparation method thereof.
Background
Tumors have become an important factor affecting human health, and tumor metastasis is an important cause of death of tumor patients. According to the statistics at present, the 1-year recurrence rate of newly diagnosed tumor patients in China is 60%, and the patients who die of tumor recurrence and metastasis exceed 80%. "early discovery, early diagnosis, early treatment" is an important approach to prevent tumor development and reduce postoperative recurrence and metastasis. Therefore, the method for researching early discovery, early diagnosis and early treatment of the tumor has important significance and social value.
In recent years, tumor studies targeting tumor-associated macrophages (TAMs) have become a focus. TAM is macrophage infiltrated in tumor tissue, is the most immune cell in tumor microenvironment, can secrete a plurality of cytokines, can identify and eliminate tumor cells at the initial stage of tumor occurrence, but plays a key role in the growth, invasion and metastasis of tumors along with the occurrence and development of the tumors. Since TAM is from self, it is closely related to the occurrence and development of tumor. Therefore, TAM is considered to be a substance having high affinity with tumors, which not only can kill tumors well, but also can deliver drugs to tumors with high efficiency. TAMs play a role in "twolip sword" in the development of tumors, and they can be differentiated into macrophages of the M1 or M2 type. Wherein, the M1 type has the anti-tumor effect, the macrophage highly expresses cytokines such as interleukin 1 (IL-1), interleukin 6 (IL-6) and the like, and the cytokines can assist in playing the anti-tumor function; however, M2 type macrophages mainly promote tumor cell invasion and metastasis and peripheral inflammatory response, and participate in tumor invasion, growth, angiogenesis, metastasis, immunosuppression and the like. Therefore, more and more studies have achieved efficient diagnosis and treatment of tumors using macrophages of type M2.
The chitosan is a natural polysaccharide, has good biocompatibility and degradability, has no biotoxicity, is rich in amino and hydroxyl, and is easy to modify. Therefore, chitosan nanomaterials are widely used in the field of nano drug delivery, for example, Cheng J., Zhou x., Zhou w., Wu h., Zhang x., Lu q., a Novel Magnetic Contrast Agent for Gastrointestinal Magnetic Imaging Through Oral administration, J Biomed nanotechnol., 2019,15(6),1162-71, which reports a Novel nuclear Magnetic resonance Contrast Agent, and multifunctional assessment of intestinal tract is achieved by loading Gd in chitosan nanoparticles and Oral administration, and the key technology is that chitosan can effectively adhere to the surface of intestinal tract Mucosa and be absorbed by intestinal tract Mucosa.
The gold nanorod is a representative gold nanomaterial, has the light absorption characteristic of high intensity in a near infrared region, and is a research hotspot in the fields of photoacoustic imaging, photothermal therapy and the like. For example, Jokerst J., Cole J., Van de Sompel D., Gambir S.S., Gold nanoparticles for ocular cancer detection with photoacoustic imaging and restriction analysis via Raman imaging in living mice, ACS nano., 6(11), 10366-77, which prepares Gold nanorods of different aspect ratios, enabling photoacoustic imaging of subcutaneous ovarian cancer in mice.
At present, precise diagnosis and treatment technologies are continuously appeared, and the research of the diagnosis and treatment technologies is usually focused on 3 aspects of high-sensitivity imaging, high-efficiency treatment and high-precision targeting. Wherein, the targeting technology is the key of tumor diagnosis and treatment; tumor localization and imaging are central to tumor diagnosis; improving the curative effect of traditional treatment means is the key point of clinical treatment of tumors. However, in the prior art, it is usually difficult to satisfy the above three aspects at the same time, and the targeting efficiency still needs to be improved due to liver first-pass effect and other factors.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a preparation method of a multifunctional intestinal tract diagnosis and treatment preparation.
Yet another object of the present invention is to: provides the multifunctional intestinal diagnosis and treatment preparation prepared by the method.
The purpose of the invention is realized by the following scheme:
the invention provides a preparation method of a multifunctional intestinal diagnosis and treatment preparation, which comprises the following steps:
(1) preparation of chitosan nanoparticles
Dissolving 30 mg of chitosan oligosaccharide and 17mg of Ethylene Diamine Tetraacetic Acid (EDTA) in 10mL of deionized water, adding 2-5 mg of gold nanorods, and performing ultrasonic dispersion uniformly; dropwise adding absolute ethyl alcohol under magnetic stirring until the color of the system changes into a milky color, adding 30 mu L of 25% glutaraldehyde solution for crosslinking for 4h, and centrifugally collecting nanospheres; dissolving 100 μ L of the target antibody in 10mL of deionized water, adding 12mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 8mg of N-hydroxysuccinimide (NHS) to activate the carboxyl group of the antibody at room temperature for 2-4 h; adding 10-15mg of nanospheres into the reaction system, stirring for 4h, centrifuging and collecting, and washing with deionized water for three times to remove unreacted antibodies to obtain a product;
(2) preparation of fluorine-containing oxygen-carrying emulsion
Adding 1-50% of fluorocarbon compound into 10ml of solvent of salt solution/ethanol with the volume ratio of 1:0.5-1:2,181, 2-dipalmitin of F: (18F-DP) and mannose-modified distearoyl phosphatidyl ethanolamine (Man-DSPE), and adding emulsifier and other auxiliary agents (the mass concentration is 0% -10% respectively), intermittently stirring at a certain speed by a homogenizer at room temperature, cooling, centrifuging, and taking supernatant to obtain fluorine-containing oxygen-carrying emulsion;
(3) macrophage-entrapped chitosan nanoparticle and fluorine-containing oxygen-carrying emulsion
And (3) incubating intestinal M2 type macrophages (with the cell density of 5000 per pore plate) with the prepared nanoparticles and the fluorine-containing oxygen-carrying emulsion for 12-72 hours to obtain the macrophage-entrapped chitosan nanoparticles and the fluorine-containing oxygen-carrying emulsion, so as to realize the multifunctional intestinal diagnosis and treatment preparation.
The diameter of the gold nanorod is 40-100 nm, and the length-diameter ratio is 2-5.
The target Antibody is one of a 5-hydroxytryptamine receptor 3 Antibody (Anti-5-HT3R), a vascular endothelial growth factor Antibody (Anti-VEGF), a histamine receptor H1 Antibody (Anti-HRH 1) and a Tryptase Polyclonal Antibody (Polyclonal Antibody to trypsin).
The fluorocarbon is one of perfluor cyclic ether, perfluor decalin, perfluor methyl cyclohexyl piperidine, perfluor bromo octane, 1-bromo pentadecafluoro heptane and 1-bromo tridecafluoro hexane.
The salt solution is one of physiological saline, phosphate buffer, acetate buffer, methylene triamcinolone, barbital buffer, sodium formate buffer, acetic acid-lithium salt buffer, acetic acid-sodium acetate buffer, acetic acid-potassium acetate buffer and other inorganic salt solutions.
The emulsifier is phospholipid including soybean lecithin, yolk lecithin, hydrogenated soybean phosphatidylcholine, hydrogenated egg phosphatidylcholine, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, distearoyl phosphatidylcholine, 1-myristoyl-2-palmitoyl phosphatidylcholine, 1-palmitoyl-2-stearoyl phosphatidylcholine, 1-stearoyl-2-palmitoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine, 1-stearoyl-2-linoleoyl phosphatidylcholine or dioleoyl phosphatidylcholine, hydrogenated dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, distearoyl phosphatidic acid, hydrogenated egg phosphatidylcholine, distearoyl phosphatidylcholine, hydrogenated egg phosphatidylcholine, distearoyl phosphatidylcholine, hydrogenated egg phosphatidylcholine, distearoyl phosphatidylcholine, di-palmitoyl phosphatidylcholine, hydrogenated egg lecithin, hydrogenated egg phosphatidylcholine, distearoyl phosphatidylcholine, hydrogenated egg yolk phosphatidylcholine, hydrogenated egg yolk phosphatidylcholine, hydrogenated egg, At least one of phaseolus vulgaris L phosphatidyl glycol amine, dipalmitoyl phosphatide L-glycol amine, cephalic phosphatidylserine, dimyristoyl county phosphatidylserine, parapalmitoyl phosphatidylserine, egg phosphatidylglycerol, dilauroyl phosphatidylglycerol, dicouyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, and cephalin.
The auxiliary agent is one or a mixture of more of Tu-80 (polyoxyethylene sorbitan monooleate), 6501 (coconut oil fatty acid diethanolamide), AEO-9 (fatty alcohol polyoxyethylene ether), Brij-35 (polyoxyethylene lauryl ether) or Triton X-100 (polyoxyethylene octyl phenyl ether).
The intermittent stirring is 1 minute, the termination is 1 minute, and the stirring time is 2-4 hours.
The invention also provides a multifunctional intestinal diagnosis and treatment preparation prepared by the method, which has a multifunctional probe for Positron Emission Tomography (PET), ultrasonic imaging and photothermal therapy, can simultaneously meet the requirements of accurate positioning and high-sensitivity long-period imaging of tumors, and improves the targeted imaging efficiency of the probe to the tumors.
The multifunctional intestinal tract diagnosis and treatment preparation is characterized by comprising a multifunctional probe with PET imaging, ultrasonic imaging and photothermal treatment, can simultaneously meet the requirements of accurate positioning and high-sensitivity long-period imaging of tumors, and improves the targeting imaging efficiency of the probe to the tumors.
The invention has the advantages that: macrophage is adopted as a drug carrier reservoir, the enrichment of the carrier at a tumor part is enhanced through the transfer effect of the macrophage, and the chitosan can be effectively adsorbed on the surface of intestinal mucosa;18the F-type nuclein is used as a PET imaging agent, and the tumor position and the metastasis are determined through PET imaging, so that the accurate positioning of the whole body tumor is realized; fluorocarbon compounds are used as ultrasound contrast agents. The diagnostic capability of local tumors is improved through ultrasonic imaging. The gold nanorods have photothermal effect and have application prospect in photothermal treatment of tumor parts.
Detailed Description
The technical solution of the present invention is further described below by specific examples. The following examples are further illustrative of the present invention and do not limit the scope of the present invention.
Example 1
A multifunctional intestinal diagnosis and treatment preparation is prepared by the following steps:
(1) preparation of chitosan nanoparticles
Dissolving 30 mg of chitosan oligosaccharide and 17mg of Ethylene Diamine Tetraacetic Acid (EDTA) in 10mL of deionized water, adding 2mg of gold nanorods, and performing ultrasonic dispersion uniformly; dropwise adding absolute ethyl alcohol under magnetic stirring until the color of the system changes into a milky color, adding 30 mu L of 25% glutaraldehyde solution for crosslinking for 4h, and centrifugally collecting nanospheres; dissolving 100 mu L of Anti-5-HT3R as a targeting antibody in 10mL of deionized water, and adding 12mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 8mg of N-hydroxysuccinimide (NHS) to activate the 3 carboxyl group of the 5-hydroxytryptamine receptor at room temperature for 2-4 h; adding 10-15mg of nanospheres into the reaction system, stirring for 4h, centrifuging and collecting, and washing with deionized water for three times to remove unreacted antibodies to obtain a chitosan nanoparticle product;
(2) preparation of fluorine-containing oxygen-carrying emulsion
Adding 10% of fluorocarbon perfluorocyclic ether and 10% of fluorocarbon perfluorocyclic ether in 10ml of a solvent of saline solution and ethanol with the volume ratio of 1:1181, 2-dipalmitoyl glyceride of F18F-DP and 5% mannose-modified distearoyl phosphatidyl ethanolamine Man-DSPE, in addition, adding 5% by mass of emulsifier soybean lecithin, intermittently stirring by a homogenizer at room temperature for 1 minute, stopping for 1 minute, stirring for 2 hours, cooling, centrifuging, and taking supernatant to obtain fluorine-containing oxygen-carrying emulsion;
(3) macrophage-coated chitosan nanoparticle and/or fluorine-containing oxygen-carrying emulsion
And (3) incubating M2 type macrophages with the cell density of 5000 per pore plate in the intestinal tract and the chitosan nanoparticles and/or the fluorine-containing oxygen-carrying emulsion for 24 hours to obtain the chitosan nanoparticles and the fluorine-containing oxygen-carrying emulsion which are wrapped by the macrophages, so as to obtain the PET/ultrasonic multifunctional intestinal tract contrast agent with the targeting function.
The particle size of the prepared multifunctional intestinal diagnosis and treatment preparation is 430 nanometers, and the dissolved oxygen is 15 percent. Under 808nm laser irradiation, 200. mu.g/mL of the product was warmed to 45 ℃ within 1 min.
Example 2
The multifunctional intestinal diagnosis and treatment preparation is similar to the step of the embodiment 1, and is prepared by the following steps:
(1) preparation of chitosan nanoparticles
Dissolving 30 mg of chitosan oligosaccharide and 10mg of tetraethyl ammonium oxalate (EDTA) in 10mL of deionized water, adding 5mg of gold nanorods, and performing ultrasonic dispersion uniformly; dropwise adding absolute ethyl alcohol under magnetic stirring until the color of the system changes into a milky color, adding 30 mu L of 25% glutaraldehyde solution for crosslinking for 4h, and centrifugally collecting nanospheres; dissolving 100 μ L of targeting antibody Anti-HRH1 in 10mL of deionized water, adding 12mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 8mg of N-hydroxysuccinimide (NHS) to activate antibody histamine receptor H1 carboxyl for 2-4H at room temperature; adding 10mg of nanospheres into the reaction system, stirring for 4h, centrifuging and collecting, and washing with deionized water for three times to remove unreacted antibodies to obtain a chitosan nanoparticle product;
(2) preparation of fluorine-containing oxygen-carrying emulsion
Adding 50% fluorocarbon perfluorodecalin and 20% fluorocarbon perfluorodecalin into 10ml of a solvent of sodium formate buffer solution/ethanol with the volume ratio of 1:2181, 2-dipalmitoyl glyceride of F18F-DP and 10% mannose-modified distearoyl phosphatidyl ethanolamine Man-DSPE, in addition, adding 10% dioleoyl phosphatidyl choline and 2% Tu-80 by mass concentration, intermittently stirring by a homogenizer at room temperature for 1 minute, stopping for 1 minute, stirring for 4 hours, cooling, centrifuging, and taking supernatant to obtain the fluorine-containing oxygen-carrying emulsion;
(3) macrophage-coated chitosan nanoparticle and/or fluorine-containing oxygen-carrying emulsion
And (3) incubating M2 type macrophages with the cell density of 5000 per pore plate in the intestinal tract and the chitosan nanoparticles and/or the fluorine-containing oxygen-carrying emulsion for 48 hours to obtain the chitosan nanoparticles and the fluorine-containing oxygen-carrying emulsion which are wrapped by the macrophages, thus obtaining the PET/ultrasonic multifunctional intestinal tract contrast agent with the targeting function.
The prepared multifunctional intestinal diagnosis and treatment preparation has the particle size of 310 nanometers and the dissolved oxygen amount of 35 percent. Under 808nm laser irradiation, 200. mu.g/mL of the product was warmed to 48 ℃ within 1 min.
Example 3
The multifunctional intestinal diagnosis and treatment preparation is similar to the step of the embodiment 1, and is prepared by the following steps:
(1) preparation of chitosan nanoparticles
Dissolving 30 mg of chitosan oligosaccharide and 17mg of Ethylene Diamine Tetraacetic Acid (EDTA) in 10mL of deionized water, adding 10mg of gold nanorods, and performing ultrasonic dispersion uniformly; dropwise adding absolute ethyl alcohol under magnetic stirring until the color of the system changes into a milky color, adding 30 mu L of 25% glutaraldehyde solution for crosslinking for 4h, and centrifugally collecting nanospheres; dissolving 100 mu L of targeting antibody Anti-VEGF in 10mL of deionized water, adding 12mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 8mg of N-hydroxysuccinimide (NHS) to activate the carboxyl of the antibody vascular endothelial growth factor at room temperature for 2-4 h; adding 15mg of nanospheres into the reaction system, stirring for 4h, centrifuging and collecting, and washing with deionized water for three times to remove unreacted antibodies to obtain a chitosan nanoparticle product;
(2) preparation of fluorine-containing oxygen-carrying emulsion
Adding 50 percent of fluorocarbon perfluorooctyl bromide and 30 percent of fluorocarbon perfluorooctyl bromide into 10ml of a solvent of saline solution acetic acid-sodium acetate buffer solution/ethanol with the volume ratio of 1:2181, 2-dipalmitoyl glyceride of F18F-DP and 20% mannose-modified distearoyl phosphatidyl ethanolamine Man-DSPE, in addition, adding 10% of fleshy pea acyl phosphatidyl glycerol and 5% of Brij-35 by mass concentration, intermittently stirring by a homogenizer at room temperature for 1 minute, stopping for 1 minute, stirring for 4 hours, cooling, centrifuging, and taking supernatant to obtain the fluorine-containing oxygen-carrying emulsion;
(3) macrophage-coated chitosan nanoparticle and/or fluorine-containing oxygen-carrying emulsion
And (3) incubating M2 type macrophages with the cell density of 5000 per pore plate in the intestinal tract and the chitosan nanoparticles and/or the fluorine-containing oxygen-carrying emulsion for 72 hours to obtain the chitosan nanoparticles and the fluorine-containing oxygen-carrying emulsion which are wrapped by the macrophages, thus obtaining the PET/ultrasonic multifunctional intestinal tract contrast agent with the targeting function.
The prepared contrast agent has the particle size of 460 nanometers and the dissolved oxygen amount of 50 percent. Under 808nm laser irradiation, 200. mu.g/mL of the product was warmed to 52 ℃ within 1 min.
Claims (10)
1. The preparation method of the multifunctional intestinal tract diagnosis and treatment preparation is characterized by comprising the following steps:
(1) preparation of chitosan nanoparticles
Dissolving 30 mg of chitosan oligosaccharide and 17mg of Ethylene Diamine Tetraacetic Acid (EDTA) in 10mL of deionized water, adding 2-5 mg of gold nanorods, and performing ultrasonic dispersion uniformly; dropwise adding absolute ethyl alcohol under magnetic stirring until the color of the system changes into a milky color, adding 30 mu L of 25% glutaraldehyde solution for crosslinking for 4h, and centrifugally collecting nanospheres; dissolving 100 μ L of the target antibody in 10mL of deionized water, adding 12mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 8mg of N-hydroxysuccinimide (NHS) to activate the carboxyl group of the antibody at room temperature for 2-4 h; adding 10-15mg of nanospheres into the reaction system, stirring for 4h, centrifuging and collecting, and washing with deionized water for three times to remove unreacted antibodies to obtain a chitosan nanoparticle product;
(2) preparation of fluorine-containing oxygen-carrying emulsion
Adding 1-50% of fluorocarbon compound into 10ml of solvent of salt solution/ethanol with the volume ratio of 1:0.5-1:2,181, 2-dipalmitoyl glyceride of F18F-DP and mannose-modified distearoyl phosphatidyl ethanolamine Man-DSPE, in addition, adding an emulsifier and/or other additives with the mass concentration of 0-10 percent respectively, intermittently stirring at a certain speed by a homogenizer at room temperature, cooling, centrifuging, and taking supernatant to obtain the fluorine-containing oxygen-carrying emulsion;
(3) macrophage-coated chitosan nanoparticle and/or fluorine-containing oxygen-carrying emulsion
And (3) incubating M2 type macrophages with the cell density of 5000 per pore plate in the intestinal tract and the chitosan nanoparticles and/or the fluorine-containing oxygen-carrying emulsion for 12-72 hours to obtain the chitosan nanoparticles and the fluorine-containing oxygen-carrying emulsion wrapped by the macrophages, so as to realize the multifunctional intestinal tract diagnosis and treatment preparation.
2. The method for preparing a multifunctional intestinal diagnosis and treatment preparation according to claim 1, wherein the gold nanorods have a diameter of 40-100 nm and an aspect ratio of 2-5.
3. The method for preparing a multifunctional intestinal diagnosis and treatment preparation according to claim 1, wherein the targeting Antibody is one of a 5-hydroxytryptamine receptor 3 Antibody Anti-5-HT3R, a vascular endothelial growth factor Antibody Anti-VEGF, a histamine receptor H1 Antibody Anti-HRH1, and a Tryptase Polyclonal Antibody to trypsin.
4. The method for preparing a multifunctional intestinal tract diagnosis and treatment preparation according to claim 1, wherein the fluorocarbon is one of perfluorocyclic ether, perfluorodecalin, perfluoromethylcyclohexylpiperidine, perfluorooctylbromide, 1-bromopentadecafluoroheptane, and 1-bromotridecafluorohexane;
the salt solution is one of physiological saline, phosphate buffer, acetate buffer, methylene triamcinolone, barbital buffer, sodium formate buffer, acetic acid-lithium salt buffer, acetic acid-sodium acetate buffer, acetic acid-potassium acetate buffer and other inorganic salt solutions.
5. The method for preparing a multifunctional intestinal diagnostic preparation according to claim 1, wherein said emulsifier is a phospholipid selected from the group consisting of soybean lecithin, egg yolk lecithin, hydrogenated soybean phosphatidylcholine, hydrogenated egg phosphatidylcholine, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, distearoyl phosphatidylcholine, 1-myristoyl-2-palmitoyl phosphatidylcholine, 1-palmitoyl-2-stearoyl phosphatidylcholine, 1-stearoyl-2-palmitoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine, 1-stearoyl-2-linoleoyl phosphatidylcholine and dioleoyl phosphatidylcholine, hydrogenated dipalmitoyl phosphatidylcholine, hydrogenated phosphatidylcholine, and mixtures thereof, At least one of distearoyl phosphatidylcholine, dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, distearoyl phosphatidic acid, phaseolus vulgaris L phosphatidyl glycol amine, dipalmitoyl phosphatidic acid glycol amine, cephalitoyl phosphatidylserine, dimyristoyl phosphatidylserine, cephalitoyl phosphatidylserine, egg phosphatidylglycerol, dilauroyl phosphatidylglycerol, dicumoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, and cephalin;
the auxiliary agent is one or a mixture of more of polyoxyethylene sorbitan monooleate Tu-80, coconut oil fatty acid diethanolamide 6501, fatty alcohol polyoxyethylene ether AEO-9, polyoxyethylene lauryl ether Brij-35 or polyethylene glycol octyl phenyl ether Triton X-100.
6. The method for preparing a multifunctional intestinal diagnosis and treatment preparation according to claim 1, wherein the intermittent stirring is 1 minute of stirring, and is stopped for 1 minute, and the stirring time is 2-4 hours.
7. The method for preparing a multifunctional intestinal diagnostic preparation according to any one of claims 1 to 6, which comprises the steps of:
(1) preparation of chitosan nanoparticles
Dissolving 30 mg of chitosan oligosaccharide and 17mg of Ethylene Diamine Tetraacetic Acid (EDTA) in 10mL of deionized water, adding 2mg of gold nanorods, and performing ultrasonic dispersion uniformly; dropwise adding absolute ethyl alcohol under magnetic stirring until the color of the system changes into a milky color, adding 30 mu L of 25% glutaraldehyde solution for crosslinking for 4h, and centrifugally collecting nanospheres; dissolving 100 mu L of Anti-5-HT3R as a targeting antibody in 10mL of deionized water, and adding 12mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 8mg of N-hydroxysuccinimide (NHS) to activate the 3 carboxyl group of the 5-hydroxytryptamine receptor at room temperature for 2-4 h; adding 10-15mg of nanospheres into the reaction system, stirring for 4h, centrifuging and collecting, and washing with deionized water for three times to remove unreacted antibodies to obtain a chitosan nanoparticle product;
(2) preparation of fluorine-containing oxygen-carrying emulsion
Adding 10% of fluorocarbon perfluorocyclic ether and 10% of fluorocarbon perfluorocyclic ether in 10ml of a solvent of saline solution and ethanol with the volume ratio of 1:1181, 2-dipalmitoyl glyceride of F18F-DP and 5% mannose-modified distearoyl phosphatidyl ethanolamine Man-DSPE, in addition, adding 5% by mass of emulsifier soybean lecithin, intermittently stirring by a homogenizer at room temperature for 1 minute, stopping for 1 minute, stirring for 2 hours, cooling, centrifuging, and taking supernatant to obtain fluorine-containing oxygen-carrying emulsion;
(3) macrophage-coated chitosan nanoparticle and/or fluorine-containing oxygen-carrying emulsion
And (3) incubating M2 type macrophages with the cell density of 5000 per pore plate in the intestinal tract and the chitosan nanoparticles and/or the fluorine-containing oxygen-carrying emulsion for 24 hours to obtain the chitosan nanoparticles and the fluorine-containing oxygen-carrying emulsion which are wrapped by the macrophages, so as to obtain the PET/ultrasonic multifunctional intestinal tract contrast agent with the targeting function.
8. The method for preparing a multifunctional intestinal diagnostic preparation according to any one of claims 1 to 6, which comprises the steps of:
(1) preparation of chitosan nanoparticles
Dissolving 30 mg of chitosan oligosaccharide and 10mg of tetraethyl ammonium oxalate (EDTA) in 10mL of deionized water, adding 5mg of gold nanorods, and performing ultrasonic dispersion uniformly; dropwise adding absolute ethyl alcohol under magnetic stirring until the color of the system changes into a milky color, adding 30 mu L of 25% glutaraldehyde solution for crosslinking for 4h, and centrifugally collecting nanospheres; dissolving 100 μ L of targeting antibody Anti-HRH1 in 10mL of deionized water, adding 12mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 8mg of N-hydroxysuccinimide (NHS) to activate antibody histamine receptor H1 carboxyl for 2-4H at room temperature; adding 10mg of nanospheres into the reaction system, stirring for 4h, centrifuging and collecting, and washing with deionized water for three times to remove unreacted antibodies to obtain a chitosan nanoparticle product;
(2) preparation of fluorine-containing oxygen-carrying emulsion
Adding 50% of fluorocarbon perfluorocyclic ether and 20% of fluorocarbon perfluorocyclic ether in 10ml of a solvent of a sodium formate buffer solution/ethanol with the volume ratio of 1:2181, 2-dipalmitoyl glyceride of F18F-DP and 10% mannose-modified distearoyl phosphatidyl ethanolamine Man-DSPE, in addition, adding 10% dioleoyl phosphatidyl choline and 2% Tu-80 by mass concentration, intermittently stirring by a homogenizer at room temperature for 1 minute, stopping for 1 minute, stirring for 4 hours, cooling, centrifuging, and taking supernatant to obtain the fluorine-containing oxygen-carrying emulsion;
(3) macrophage-coated chitosan nanoparticle and/or fluorine-containing oxygen-carrying emulsion
And (3) incubating M2 type macrophages with the cell density of 5000 per pore plate in the intestinal tract and the chitosan nanoparticles and/or the fluorine-containing oxygen-carrying emulsion for 48 hours to obtain the chitosan nanoparticles and the fluorine-containing oxygen-carrying emulsion which are wrapped by the macrophages, thus obtaining the PET/ultrasonic multifunctional intestinal tract contrast agent with the targeting function.
9. The method for preparing a multifunctional intestinal diagnostic preparation according to any one of claims 1 to 6, which comprises the steps of:
(1) preparation of chitosan nanoparticles
Dissolving 30 mg of chitosan oligosaccharide and 17mg of Ethylene Diamine Tetraacetic Acid (EDTA) in 10mL of deionized water, adding 10mg of gold nanorods, and performing ultrasonic dispersion uniformly; dropwise adding absolute ethyl alcohol under magnetic stirring until the color of the system changes into a milky color, adding 30 mu L of 25% glutaraldehyde solution for crosslinking for 4h, and centrifugally collecting nanospheres; dissolving 100 mu L of targeting antibody Anti-VEGF in 10mL of deionized water, adding 12mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 8mg of N-hydroxysuccinimide (NHS) to activate the carboxyl of the antibody vascular endothelial growth factor at room temperature for 2-4 h; adding 15mg of nanospheres into the reaction system, stirring for 4h, centrifuging and collecting, and washing with deionized water for three times to remove unreacted antibodies to obtain a chitosan nanoparticle product;
(2) preparation of fluorine-containing oxygen-carrying emulsion
Adding 50 percent of fluorocarbon perfluorooctyl bromide and 30 percent of fluorocarbon perfluorooctyl bromide into 10ml of a solvent of saline solution acetic acid-sodium acetate buffer solution/ethanol with the volume ratio of 1:2181, 2-dipalmitoyl glyceride of F18F-DP and 20% mannose-modified distearoyl phosphatidyl ethanolamine Man-DSPE, in addition, adding 10% of fleshy pea acyl phosphatidyl glycerol and 5% of Brij-35 by mass concentration, intermittently stirring by a homogenizer at room temperature for 1 minute, stopping for 1 minute, stirring for 4 hours, cooling, centrifuging, and taking supernatant to obtain the fluorine-containing oxygen-carrying emulsion;
(3) macrophage-coated chitosan nanoparticle and/or fluorine-containing oxygen-carrying emulsion
And (3) incubating M2 type macrophages with the cell density of 5000 per pore plate in the intestinal tract and the chitosan nanoparticles and/or the fluorine-containing oxygen-carrying emulsion for 72 hours to obtain the chitosan nanoparticles and the fluorine-containing oxygen-carrying emulsion which are wrapped by the macrophages, thus obtaining the PET/ultrasonic multifunctional intestinal tract contrast agent with the targeting function.
The prepared contrast agent has the particle size of 460 nanometers and the dissolved oxygen amount of 50 percent. Under 808nm laser irradiation, 200. mu.g/mL of the product was warmed to 52 ℃ within 1 min.
10. A multifunctional intestinal tract diagnosis and treatment preparation, which is prepared according to the method of any one of claims 1 to 9, is provided with a multifunctional probe for PET imaging, ultrasonic imaging and photothermal therapy, and can simultaneously meet the requirements of accurate tumor positioning and high-sensitivity long-period imaging.
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| CN202111620713.4A CN114306276B (en) | 2021-12-28 | 2021-12-28 | Preparation method of multifunctional intestinal diagnosis and treatment preparation and product thereof |
| PCT/CN2021/143578 WO2022179307A1 (en) | 2021-02-24 | 2021-12-31 | Method for preparing multifunctional intestinal diagnosis and treatment formulation, and product thereof |
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| CN202111620713.4A CN114306276B (en) | 2021-12-28 | 2021-12-28 | Preparation method of multifunctional intestinal diagnosis and treatment preparation and product thereof |
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